RNAi-mediated silencing of lysosomal ASM inhibits host cell invasion by T. cruzi, and exposure to extracellular sphingomyelinase restores parasite entry. (A) Immunoblot with anti-ASM antibodies in extracts of HeLa cells treated with control or ASM siRNA. The arrow points to the band corresponding to ASM; the higher additional band is an unspecific reaction. Immunoblot was probed with anti-actin antibodies for loading control. (B) Quantification of trypomastigote invasion in siRNA-treated HeLa cells. After siRNA treatment, cells were incubated with trypomastigotes for the indicated periods, fixed, and processed for quantification of intracellular parasites. The data correspond to the mean of triplicates ± SD. **, P = 0.0073, Student’s t test; ***, P < 0.0001, Student’s t test comparing control (gray bars) and ASM siRNA-treated cells (black bars). (C) siRNA-treated HeLa cells were incubated with trypomastigotes in the presence or absence of the indicated concentrations of bacterial sphingomyelinase for 30 min, fixed, and processed for quantification of intracellular parasites. The data correspond to the mean of triplicates ± SD. **, P < 0.0014; ***, P = 0.0003, Student’s t test comparing control (gray bars) and treated cells (black bars). These results are representative of four independent experiments.